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Abstract:

The present invention is directed to devices, systems and methods that
enable the detection of low copy numbers of bacterial polynucleotides in
a sample without having to use multiple species specific primer
sequences.

Claims:

1. A probe for detecting target nucleic acid material in a sample, the
probe comprising: a universal probe sequence hybridizable to a target
sequence; a unique primer sequence interconnected to the probe sequence;
and a solid-phase medium associated with said probe sequence and unique
primer sequence.

2. The probe of claim 1, wherein in the unique primer sequence is
engineered to avoid binding with non-target nucleic acid material or
contaminants in the sample.

3. The probe of claim 1, wherein the target nucleic acid material is a
polynucleotide from an organism or virus.

4. The probe of claim 2, wherein the target nucleic acid material is
bacterial, fungal, viral, or any other infectious agent.

5. The probe of claim 1, wherein the unique primer sequence is adjacent
to the probe sequence.

6. The probe of claim 1 where in the unique primer sequence is engineered
to avoid binding with non-target nucleic acid material or contaminants in
the sample.

7. A probe for detecting bacterial nucleic acid material in a sample, the
probe comprising: a universal probe sequence hybridizable with
polynucleotide sequences of multiple bacterial species; and a
non-bacterial primer sequence interconnected with said universal probe
sequence.

8. The probe of claim 7, further comprising a solid-phase medium
associated with said universal probe sequence and nonbacterial primer
sequence.

11. The probe of claim 8, wherein said solid-phase medium is a wall of a
well, dish or other container capable of holding a fluid.

12. The probe of claim 7, wherein said universal probe sequence is an RNA
or DNA sequence.

13. The probe of claim 7, wherein said universal probe sequence and said
non-bacterial primer sequence are on the same strand.

14. The probe of claim 7, wherein said non-bacterial primer sequence
comprises a sequence of at least 5, 10, 15, 20, or 25 bases that are
lacking in 10 or more natural species of bacteria.

15. The probe of claim 8, wherein said probe further comprises a spacer
sequence between the solid phase medium and the probe sequence or primer
sequence.

16. The probe of claim 15, wherein the spacer sequence is a strand of
common nucleic acid bases linked to the non-bacterial primer sequence on
one end and to biotin on the other end, and wherein said biotin is bound
to said bead.

17. The probe of claim 1, wherein said solid-phase medium is a magnetic
bead.

18. The probe of claim 1, wherein said universal probe sequence is an RNA
or DNA sequence specific to 16S RNA of multiple bacterial species.

20. A probe for detecting bacterial nucleic acid material in a sample,
the probe comprising: a probe strand comprising (i) a universal probe
sequence hybridizable with polynucleotide sequences of multiple bacterial
species, the universal probe sequence having a Tm of from 45-55.degree.
C.; and (ii) a non-bacterial primer sequence interconnected with said
universal probe sequence.

21. The probe of claim 20, further comprising an adenine strand linked
with the probe strand on one end and biotin on the other end and a
solid-phase medium comprising streptavidin bound to said biotin.

22. A method of detecting bacterial nucleic acid material in a sample,
the method comprising contacting the sample with a probe comprising (i) a
universal probe sequence hybridizable with polynucleotide sequences of
multiple bacterial species; (ii) a nonbacterial primer sequence
interconnected with said universal probe sequence; and selectively
amplifying any bacterial nucleic acid material in said sample that is
captured by said probe.

23. The method of claim 22, wherein the probe further comprises (iii) a
solid-phase medium associated with said universal probe sequence and said
non-bacterial primer sequence.

24. The method of claim 22, wherein the bacterial nucleic acid material
captured by the probe is a DNA or RNA sequence.

25. The method of claim 22, wherein the bacterial nucleic acid material
captured by the probe is an RNA sequence.

26. The method of claim 22, further comprising subjecting the RNA
sequence to reverse transcriptase under conditions to produce a DNA
extension on the same strand as the universal probe sequence, the DNA
extension being complementary to a portion of the RNA sequence not
hybridized to the universal probe sequence.

27. The method of claim 26, wherein the universal probe sequence and DNA
extension form a base strand, and wherein the method further comprises
selectively amplifying comprises conducting a polymerase chain reaction
(PCR) using said base strand.

30. The method of claim 27, wherein said PCR includes traditional PCR.

31. The method of claim 27, wherein said PCR comprises combining said
base strand, whether associated with said solid-phase medium or not, in a
reaction mixture with a first primer complimentary to said non-bacterial
primer sequence and a second primer complimentary to a sequence on said
DNA extension.

32. A method of detecting target nucleic acid material in a sample, the
method comprising contacting the sample with a probe comprising a probe
sequence hybridizable to a target sequence; a unique primer sequence
interconnected to the probe sequence; and a solid-phase medium associated
with said probe sequence and unique primer sequence; and selectively
amplifying any target nucleic acid material in said sample that is
captured by said probe.

33. The method of claim 32, wherein the target nucleic acid material is
an RNA sequence, and further comprising subjecting the captured RNA
sequence to reverse transcriptase under conditions to produce a DNA
extension on the same strand as the universal probe sequence, the DNA
extension being complementary to a portion of the RNA sequence not
hybridized to the universal probe sequence.

34. The method of claim 33, wherein the universal probe sequence and DNA
extension form a base strand and said selectively amplifying comprises
conducting a polymerase chain reaction (PCR) using said base strand.

35. The method of claim 32, further comprising blocking the probe after
capture of the RNA sequence but prior to subjecting the RNA sequence to
reverse transcriptase, and/or after subjecting the RNA sequence to
reverse transcriptase.

36. The method of claim 35, wherein said blocking comprises contacting
the probe with nucleotides.

37. The method of claim 35, wherein said blocking comprises contacting
the probe with deoxynucleotides.

38. The method of claim 35, wherein said blocking comprises contacting
the probe with deoxythymidine triphosphate.

42. A method of detecting target nucleic acid material in a sample, the
method comprising contacting the sample with a fusion primer comprising:
(a) a universal probe sequence hybridizable with polynucleotide sequences
of multiple bacterial species; and (b) a non-bacterial sequence
interconnected with said universal probe sequence; subjecting the
captured polynucleotide sequence to reverse transcriptase under
conditions to produce a base strand having a DNA extension on the same
strand as the universal probe sequence and the non-bacterial sequence;
conducting a polymerase chain reaction (PCR) using the base strand
comprising the non-bacterial sequence, the universal probe sequence and
the DNA extension; and wherein the PCR comprises: i) with the DNA strand
dissolved in a PCR reaction mixture comprising said DNA strand, primers,
a DNA polymerase, heating said PCR reaction mixture sufficiently to
achieve denaturation of the base strand into single-strand DNA, wherein
the primers comprise a first primer complimentary to said non-bacterial
primer sequence and a second primer complimentary to a sequence on the
DNA extension; ii) cooling the PCR reaction mixture sufficiently to cause
the primers to anneal to said single-strand DNA and to elongate and
thereby at least partially form a DNA strand complementary to said
single-strand DNA; and iii) subjecting said PCR reaction mixture to a
reaction temperature of about 65.degree. C. to further elongate said
complementary DNA strand formed in step (ii); and iv) repeating steps
(i)-(iii) to define a subsequent PCR step; wherein the universal probe
sequence is rendered at least partially inoperable to participate in the
PCR at least before the subsequent PCR step.

43. The method of claim 42, wherein the universal probe sequence is
inactivated during the subjecting step.

44. The method of claim 42, wherein the temperature differential between
a Tm of the universal probe sequence and the reaction temperature in
step (iii) is effective to preventive hybridization of universal probe
sequence with contaminants in the PCR reaction mixture.

45. The method of claim 42, wherein the universal probe sequence has a
Tm that at least 10 degrees lower than the reaction temperature.

46. The method of claim 42, wherein the universal probe sequence is
engineered to bind to >90% of known bacterial isolates.

47. The method of claim 42, wherein the universal probe sequence is
removed from the PCR reaction mixture so that it cannot participate in
the subsequent PCR step.

48. The method of claim 42, wherein the universal probe sequence is
constructed in such a manner that it cannot participate in the subsequent
PCR step.

49. The method of claim 42, wherein the universal probe sequence is
shortened to the extent that it cannot form a PCR product in the
subsequent PCR step.

50. The method of claim 42, wherein the universal sequence is modified or
contains modified nucleotides such that it cannot form a PCR product in
the subsequent PCR step.

51. The method of claim 49, wherein the modified nucleotides are
effective to increase the affinity of the universal sequence for RNA.

52. The method of claim 48, wherein the modified nucleotides are
effective increase the affinity of either the non-bacterial sequence or
the universal probe sequence such that the PCR can be carried out at
sufficiently high temperature such that a PCR product comprising the
universal probe sequence from the PCR reagents is not generated.

53. The method of claim 41, further comprising diluting the PCR reaction
mixture prior to said heating in step (i) of the PCR.

54. The method of claim 41, wherein the PCR reaction is diluted with
buffer in a range of 1:20 to 1:60 by volume.

55. The method of claim 41, wherein the fusion primer is enzymatically
destroyed prior to the PCR.

Description:

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is related to U.S. Provisional Application No.
61/542,470 filed Oct. 3, 2011, U.S. Provisional Application No.
61/550,424 filed Oct. 23, 2011, and U.S. Provisional Application No.
61/655,071 filed Jun. 4, 2012 to which priority is claimed under 35 USC
119 and which are incorporated herein by reference in their entirety.

FIELD OF THE INVENTION

[0002] The present invention relates to a device, system and apparatus for
detecting bacterial infections in biological materials.

BACKGROUND OF THE INVENTION

[0003] Development of a rapid diagnostic test for detecting bacterial
infection would have a significant impact on the management of
infections. For the identification of pathogens and antibiotic resistance
genes in clinical samples, DNA probe and DNA amplification technologies
offer several advantages over conventional methods. The organism can be
detected directly in clinical samples, thereby reducing the cost and time
associated with isolation of pathogens. Also, bacterial genotypes (at the
DNA level) are more stable than the bacterial phenotypes (i.e.
biochemical properties). DNA-based technologies have proven to be
extremely useful for specific applications in the clinical microbiology
laboratory (and a method to quantify small amounts of DNA). For example,
kits for the detection of fastidious organisms based on the use of
hybridization probes or DNA amplification for the direct detection of
pathogens in clinical specimens are commercially available (Persing et
al, 1993. Diagnostic Molecular Microbiology: Principles and Applications,
American Society for Microbiology, Washington, D.C.).

[0004] The conventional DNA-based tests for the detection and
identification are based on the amplification of the highly conserved 16S
rRNA gene followed by hybridization with internal species-specific
oligonucleotides. The significance of the 16SrRNA gene is that certain
sequences are conserved in all gene variants. The subsequent
hybridization targets and allows for amplification of species-specific
oligonucleotides which are derived from species-specific bacterial
genomic DNA fragments. However, ultimately, these conventional strategies
using universal sequences suffer from the fact that the use of Taq
polymerase interferes with the detection. Contamination of the Taq
polymerase with bacterial nucleic acid was first described over 20 years
ago. See Rand and Houck, Molecular and Cellular Probes (1990) 4:445-450.
This means if one uses primers targeting areas of the 16 S ribosomal RNA
(or DNA) that are shared by many bacteria, the contamination of the Taq
becomes a limiting factor in detecting low copy numbers of bacteria. In
applying such a method to the detection of bacteria in normally sterile
clinical specimens, the Taq enzyme contamination forces the use of
primers specific to various species of bacteria, rather than allowing the
use of sequences that could amplify all or many species.

BRIEF DESCRIPTION OF THE DRAWINGS

[0005] FIGS. 1a-1e show a stepwise diagram of a probe embodiment and
method of using the probe to selectively amplify a target product.

[0006] FIG. 2 shows a gel that demonstrates how specific and sensitive the
method embodiment is at capturing and detecting bacterial nucleic acid
material in a sample.

[0007] FIG. 3 shows the temperature dependence of the false positive
product with reagents alone i.e. that if the Tm of the universal part of
the fusion primer is low enough there can be no PCR product if the PCR is
carried out at a high enough temperature.

[0008] FIG. 4a-4b show proof of principle that includes both dilution of
the RT reaction mixture (1:50) and by using a high enough annealing
temperature in the PCR (68° C. in FIG. 4; 65° C. in FIG.
5).

[0009] FIGS. 5a-5f show a stepwise diagram of a probe embodiment and
method of using the probe to selectively amplify a target product.

DETAILED DESCRIPTION OF THE INVENTION

[0010] Aspects of the present invention are directed to devices, systems
and methods that enable the detection of low copy numbers of bacterial
polynucleotides in a sample without having to use multiple species
specific primer sequences. In this way, aspects of the present invention
provide highly sensitive diagnostic tests capable of detecting
essentially all potential bacterial pathogens in a biological sample
within a short period of time, e.g., hours. In one aspect, an initial
primer is utilized comprising a non-bacterial sequence interconnected to
a universal bacterial sequence. The universal sequence is removed,
destroyed, inactivated, etc. such that it does not interfere in a PCR
step by inhibiting the PCR itself and/or by causing a false positive from
the contaminating bacterial DNA in the Taq enzyme.

[0011] In accordance with one aspect, the present invention pertains to a
probe for detecting target nucleic acid material in a sample. The target
nucleic acid material may comprise polynucleotides from any organism or
virus, including but not limited to plant and animal polynucleotides. In
one embodiment, the target nucleic acid is a bacterial, fungal, viral, or
other infectious agent. The probe may contain a universal probe sequence
hybridizable to a target sequence. There is interconnected to the probe
sequence, whether adjacent or non-adjacent, a unique primer sequence.

[0012] The unique primer sequence is engineered to have an arbitrary
sequence that hybridizes to a unique primer. Thus, the unique primer
sequence may be utilized to develop a primer for use in an amplification
step as will be explained in further detail below. In addition, the
arbitrary sequence is one that avoids undesired binding with the target
sequence or possible nucleic acid contaminants in the sample.
Contaminants would be nucleic acid sequences in the test reagents that if
amplified would interfere with detection of the target nucleic acid in a
patient (or other) sample of interest, e.g., including nucleic acids from
any organism whether bacterial, fungal, other infectious agent or even
human, animal and plant. The probe sequence and unique primer sequence
are typically on the same strand and, in certain embodiments, are
associated with a solid phase medium.

[0013] In another aspect, the probe includes a universal probe sequence
hybridizable with polynucleotide sequences of multiple bacterial species
and a non-bacterial primer sequence interconnected with the universal
probe sequence. In one embodiment, the probe may further comprise a
solid-phase medium associated with the universal probe sequence and the
non-bacterial primer sequence; alternatively the solid-phase medium may
be associated with the 2nd universal primer used in the PCR with the
unique primer. In certain embodiments, the universal probe sequence
comprises a DNA sequence or an RNA sequence. The universal probe sequence
and the non-bacterial primer sequence may be on the same strand. In one
embodiment, the non-bacterial primer sequence includes a sequence of at
least 5, 10, 15, 20, or 25 bases that are lacking in 10 or more natural
species of bacteria.

[0014] When utilized, the solid-phase medium may be any suitable medium
for binding of the universal probe so that the probe is isolated to
enhance sensitivity and yield downstream. In one embodiment, the
solid-phase medium comprises a bead. In a particular embodiment, the bead
comprises a magnetic bead. In alternative embodiments, the solid-phase
medium comprises wall of a well, dish or other container capable of
holding a fluid. The probe may further include an adenine strand linked
to the non-bacterial primer sequence on one end and to Biotin on the
other end. In this embodiment, the Biotin is typically bound to the bead.

[0015] In further embodiments, the universal probe sequence is an RNA or
DNA sequence specific to 16S RNA of multiple bacterial species. In one
embodiment, the universal probe sequence is used to target a region of
16SrRNA and to amplify the target in parts. In a particular embodiment,
the universal probe sequence is engineered to bind to >90% of known
bacterial isolates.

[0016] According to another aspect, the invention pertains to a method of
detecting bacterial nucleic acid material in a sample. The method
includes contacting the sample with a probe comprising: (i) a universal
probe sequence hybridizable with polynucleotide sequences of multiple
bacterial species; and (ii) a non-bacterial primer sequence
interconnected with the universal probe sequence. The method further
comprises selectively amplifying any bacterial nucleic acid material in
the sample that is captured by the probe. In one embodiment, the
bacterial nucleic acid material captured by the probe comprises a DNA or
an RNA sequence. In certain embodiments, the probe further comprises
(iii) a solid-phase medium associated with the universal probe sequence
and the non-bacterial primer sequence.

[0017] In accordance with another aspect, the captured nucleic acid is an
RNA sequence and the method further comprises step of subjecting the RNA
sequence to reverse transcriptase under conditions to produce a DNA
extension on the same strand as the universal probe sequence, the DNA
extension being complementary to a portion of the RNA sequence not
hybridized to the universal probe sequence. The universal probe sequence
and DNA extension may form a base strand. This strand may be made
double-stranded in one embodiment by enzymatic methods as are well-known
in the art. The method may further comprise a selectively amplifying
step, which may be a polymerase chain reaction (PCR) using the base
strand. The use of PCR may include the implementation of real-time PCR.
Further, the PCR may include combining the base strand, whether
associated with said solid-phase medium or not, in a reaction mixture
with a first primer complimentary to the non-bacterial primer sequence
and a second primer complimentary to a sequence on the DNA extension.

[0018] In accordance with another aspect, there is provided a method of
detecting target nucleic acid material in a sample. The method comprises
contacting the sample with an initial probe comprising: (a) a universal
probe sequence as described herein hybridizable with polynucleotide
sequences of multiple bacterial species; and (b) a non-bacterial sequence
interconnected with the universal probe sequence. The method comprises
subjecting a captured polynucleotide sequence to reverse transcriptase
under conditions to produce a base strand comprising a DNA extension on
the same strand as the universal probe sequence. In one embodiment, the
DNA extension is complementary to a portion of the RNA sequence not
hybridized to the universal probe sequence. This strand may be made
double-stranded in one embodiment by enzymatic methods as are well-known
in the art.

[0019] Thereafter, the method comprises conducting a polymerase chain
reaction (PCR) using a base strand comprising the non-bacterial sequence,
the universal probe sequence and the DNA extension. The PCR comprises:
with the target DNA dissolved in a PCR reaction mixture comprising the
base strand, primers, a DNA polymerase, heating said PCR reaction mixture
sufficiently to achieve denaturation of the base strand into
single-strand DNA. In this step, the primers comprise a first primer
complimentary to the non-bacterial primer sequence and a second primer
complimentary to a sequence on the DNA extension. In addition, the PCR
comprises cooling the PCR reaction mixture sufficiently to cause the
primers to anneal to the single-strand DNA and to elongate and thereby at
least partially form a DNA strand complementary to the single-strand DNA.
Further, the PCR comprises step (iii) of subjecting the PCR reaction
mixture to a reaction temperature of about 65° C. to further
elongate the complementary DNA strand formed in step (ii). Thereafter,
the PCR steps may be repeated as is known in the art as desired.

[0020] Critically, the universal probe sequence interconnected to the
universal primer sequence is rendered at least partially inoperable to
participate in the PCR before the subsequent PCR step to avoid inhibition
of the PCR or production of a false positive PCR product. For example,
the universal probe sequence may be removed, destroyed, inactivated,
diluted, or otherwise rendered non-functional etc. such that it does not
interfere in a PCR step by inhibiting the PCR itself and/or by causing a
false positive from the contaminating bacterial DNA in the Taq enzyme. It
is appreciated therefore that the initial primer may be rendered
inoperable by various methods as would be appreciated by one skilled in
the art. In one embodiment, as explained below in the Examples below, the
universal probe sequence is constructed so as to have a relatively low
Tm. In one embodiment, the universal probe sequence has a Tm of
from 45-55° C. Similarly, the non-bacterial sequence has a
relatively high Tm. In one embodiment, the non-bacterial sequence
has a sufficiently high Tm to allow for annealing in PCR at a
temperature that is at least 10° C. greater than the universal
probe sequence Tm. In one embodiment, the annealing temperature in
the PCR is from 65-70° C. rather than the standard 55-60°
C. Advantageously, because the Tm of the universal portion is so
low, it can never hybridize with the contaminating DNA in the Taq enzyme
during the PCR because the lowest temperature in the PCR remains
10° C. greater than the universal probe sequence Tm.

[0021] In one embodiment, the universal probe sequence is removed from the
PCR reaction mixture so that it cannot participate in the subsequent PCR
step. In this instance, the universal probe is removed by enzymatic
digestion, or by physico-chemical means, or even sufficiently diluted. In
another embodiment, the universal probe sequence is constructed in such a
manner that it cannot participate in the subsequent PCR step. For
example, the universal probe sequence may be shortened to the extent that
it cannot form a PCR product in a subsequent PCR step. In another
embodiment, the universal sequence is modified or contains modified
nucleotides such that it cannot form a PCR product in the subsequent PCR
step. In one embodiment, the modified nucleotides may be effective to
increase the affinity of the universal sequence for RNA.

[0022] In another aspect, embodiments of the PCR method described herein
further comprise diluting the PCR reaction mixture prior to the heating
step of the PCR. In one embodiment, the PCR reaction mixture is diluted
with a suitable medium, e.g., buffer, in a range of 1:20 to 1:60 by
volume. Without wishing to be bound by theory, it is believed that the
dilution of the PCR reaction mixture aids in increasing the signal to
noise ratio in a subsequent detection step following PCR.

[0024] Turning now to the drawings, FIG. 1 depicts a stepwise method
showing how bacterial nucleic acid material can be selectively captured
and amplified, which in turn enables the identification of bacterial
infection. This identification can occur even when there is a low copy
number in the sample. FIG. 1 a shows a probe 100 that includes a specific
probe sequence 102 that is universal to several bacterial species; thus,
it may also be referred to as a "universal probe sequence" or "universal
sequence." The probe 100 also includes a unique sequence 104 on the same
strand as the universal probe sequence 102. The unique sequence 104 is
specifically designed to lack bacterial sequences, and typically pertains
to at least 5, 10, 15, 20, or 25 bases. The unique sequence 104 may also
be referred to as a non-bacterial sequence. The non-bacterial sequence
104 typically lacks homology or does not recognize bacterial sequences
from at least 10 or more bacterial species.

[0025] The probe 100 further includes a linker sequence 106 adjacent to
the non-bacterial sequence 104. The linker sequence 106 links to a biotin
molecule 108. In one embodiment, the linker sequence 106 may comprise a
series of adenine bases. The biotin 108 binds to a streptoavidin molecule
112 bound to a solid phase substrate 110. The solid phase substrate 110
shown is a magnetic bead, but it is understood that the present invention
is not so limited. In operation, the probe 100 is exposed to a sample,
such as, but not limited to, a biological fluid (blood, mucous, vaginal
fluid, serum, semen etc.), tissue sample (typically a tissue sample
expected of being infected, and may be homogenized), food sample, or any
liquid or other sample (including nucleic acid extracts thereof)
suspected of being infected with a bacterium. Typically, the sample is
suspected to contain both human and bacterial RNA. RNA 130 in the sample
hybridizes to the universal probe sequence 104 at a complimentary
sequence 132 (FIG. 1 b). By isolating the bead 110, the captured RNA 130
is washed of non-bound nucleic acid.

[0026] The resulting DNA-RNA hybrid 134 attached to the bead 110 is used
as the primer/template for reverse transcriptase (RT) to copy the
hybridized bacterial RNA thereby forming a cDNA extension strand 140
(FIG. 1c). The bead 110 is then washed again. Using a PCR primer 150
likely to bind to a site downstream on the cDNA extension strand and a
primer 104' directed to the non-bacterial sequence 104 (FIG. 1d), PCR is
further conducted (FIG. 1e) to amplify the target product to produce
product 160. Even though universal bacterial sequences were used to
capture the RNA and a bacterial universal primer was used as the PCR
primer 150, the non-bacterial sequence 104 allows for completely specific
amplification of the RNA that has been captured and copied. It is noted
that regular PCR or real-time PCR (rtPCR), can be conducted to amplify
the target sequences. rtPCR provides a more rapid means of detecting the
presence of bacterial nucleic acid material in a sample.

[0027] In accordance with another aspect, as shown in FIG. 5, the
unique-universal probe 100 comprising universal sequence 102 and unique
sequence 104 is not attached to a solid phase, but is instead allowed to
function as a primer for the reverse transcriptase (RT)(Fig. 5a). RNA 130
in the sample hybridizes to the universal probe sequence 104 at a
complimentary sequence 132 (FIG. 5b). The resulting hybrid 134 is used as
the primer/template for reverse transcriptase (RT) to copy the hybridized
bacterial RNA, thereby forming a cDNA extension strand 140 (FIG. 5c).
After RT, a second strand 170 is made using Klenow DNA Polymerase and a
2nd universal primer 180 that has been synthesized with a biotin 108
on its 5' end (FIG. 5d). After the Klenow step, the double-stranded
product 190 can now attach to a streptavidin molecule 112 on the solid
phase substrate 110 (FIG. 5e). In this embodiment, the solid phase
substrate 110 comprises magnetic beads. After washing, the solid phase
substrate 110 with attached product 190 can be treated enzymatically or
in any other way to remove any residual universal-unique probe 100 prior
to PCR. PCR is then conducted to amplify the double-stranded product 190
to produce product 200 (FIG. 5f).

[0028] Referring to FIG. 2, FIG. 2 shows a gel where various samples were
used to demonstrate the selectivity of the method embodiments. As shown,
lanes 3 and 4, which were known to have bacterial infection, show a clear
band of a specific molecular weight related to the bacterial PCR primer
chosen illustrating the amplified target product. In addition, the
inventors have discovered that commercially available reverse
transcriptase is actually contaminated with nucleic acid sequences.
Moreover, these contaminating sequences can interfere with the detection
of nucleic acids according to the methods described herein. Accordingly,
in a specific embodiment, reverse transcriptase is enzymatically treated
prior to use to clean it of these contaminating sequences. Thus, one
aspect of the invention pertains to nucleic acid-free reverse
transcriptase. Enzymes used for this purpose include endonuclease(s). The
cleaned reverse transcriptase, or nucleic acid-free reverse
transcriptase, is then used in the process as described above. Any other
enzymes used prior to the PCR, for example, Klenow reagent to make the
reverse transcriptase cDNA product double-stranded, are likewise rendered
non-contaminated.

[0029] In another aspect, the probe can be blocked upon capture of target
nucleic acid material. This would be done after subjecting the probe to
reverse transcriptase.

[0030] Nucleotides would typically be used to block the remaining probe
not extended by reverse transcriptase. In a more specific embodiment, the
nucleotides are deoxynucleotides. In a specific example, deoxythymidine
triphosphate, or a similar deoxynucleotide is used to block the probe.

EXAMPLES

Example 1

[0031] After reverse transcription, the reaction mixture was diluted
significantly (generally in the range of 1:20-1:60). Because the fusion
primer not only can give a false positive from reagents alone, but can
also interfere with the sensitivity of the PCR itself, the fusion primer
was designed that the universal sequence portion is relatively short and
the unique (non-bacterial) sequence is relatively long. By lengthening
the universal part, the melting temperature (Tm) of the primer is
reduced down to the range of 45-55° C. thus reducing its binding
affinity. By lengthening the unique portion, the Tm is raised thus
raising the temperature of the PCR to an annealing temperature of
65-70° C. as opposed to the standard 55-60° C. One of the
critical aspects of the present invention is that the universal sequence
does not hybridize with the contaminating DNA in the Taq enzyme during
the PCR because the Tm of the universal sequence is more than
10° C. lower than the lowest temperature in the PCR. In order to
ensure the second universal primer does hybridize, its Tm remained
high by lengthening it without losing its universality characteristics.
Modified nucleotides, for example, may be used that raise the Tm of
a primer into which they have been incorporated Proof of principle was
demonstrated of both the need and effectiveness of dilution, as well as
the effectiveness of lowering the Tm of the universal portion of the
fusion primer while raising the Tm of the unique portion and its
corresponding 2nd PCR primer.

[0032] FIG. 3 shows the temperature dependence of the false positive
product with reagents alone, namely that if the Tm of the universal
part of the fusion primer is low enough, there can be no false positive
PCR product if the PCR is carried out at a high enough temperature. FIGS.
4-5 show proof of principle that includes both dilution of the RT
reaction mixture (1:50) and by using a high enough annealing temperature
in the PCR. Both full sensitivity (in this case approximately 200
copies/reaction mixture) and no false positives were obtained at
annealing temperatures of 65° C. and 68° C.

Example 2

[0033] Pseudomonas aeruginosa RNA was diluted using a preparation that
corresponds to approximately 200 copies/reaction mixture at 1:100M. The
lanes that are labeled in FIGS. 3,-4a, and 4b correspond to an
approximately 230 bp product while the unlabeled lanes are from a longer
PCR product of around 450 bp. The reaction is significantly more
sensitive when carrying out the shorter PCR. The reverse transcriptase
reaction mixture was diluted 1:50 before performing the PCR.

[0034] It should be borne in mind that all patents, patent applications,
patent publications, technical publications, scientific publications, and
other references referenced herein and in the accompanying appendices are
hereby incorporated by reference in this application to the extent not
inconsistent with the teachings herein. It is important to an
understanding of the present invention to note that all technical and
scientific terms used herein, unless defined herein, are intended to have
the same meaning as commonly understood by one of ordinary skill in the
art. The techniques employed herein are also those that are known to one
of ordinary skill in the art, unless stated otherwise. For purposes of
more clearly facilitating an understanding the invention as disclosed and
claimed herein, the following definitions are provided.

[0035] While a number of embodiments of the present invention have been
shown and described herein in the present context, such embodiments are
provided by way of example only, and not of limitation. Numerous
variations, changes and substitutions will occur to those skilled in the
art without materially departing from the invention herein.

[0036] For example, the present invention need not be limited to best mode
disclosed herein, since other applications can equally benefit from the
teachings of the present invention. Also, in the claims,
means-plus-function and step-plus-function clauses are intended to cover
the structures and acts, respectively, described herein as performing the
recited function and not only structural equivalents or act equivalents,
but also equivalent structures or equivalent acts, respectively.
Accordingly, all such modifications are intended to be included within
the scope of this invention as defined in the following claims, in
accordance with relevant law as to their interpretation.